The aim of our study was to advance the knowledge about the biological differences in the healing of the anterior cruciate ligament (ACL) versus the medial collateral ligament (MCL). We quantified α-smooth muscle actin (α-SMA) expression and TGF-β receptor I (TGF-βRI) expression in experimentally injured rabbit ligaments (from day 3 to 12 weeks post-injury). Myofibroblasts (α-SMA positive cells) were identified as early as the third day post-injury in MCL and their density increased steadily up to day 21. Myofibroblasts were also detected in injured ACL but their density remained very low at all time points. The percentage of positive TGF-βRI area significantly increased in both injured ligaments compared to controls, with a peak expression at day 21; however, it remained constantly lower in ACL compared to MCL. A significant correlation was found between the percentage of TGF-βRI positive cells and the percentage of α-SMA expression only in injured MCL. These results provide evidence that myofibroblasts are important players in MCL remodelling after injury. The combined presence of myofibroblasts and TGF-βRI in the first 3 weeks post-MCL injury may partially explain the difference in the MCL and ACL healing process.